1. Field of the Invention
A high yield method for producing ethanol from carbohydrate fermentation, by treating incoming carbohydrate material with an aldehyde, a fatty acid, a terpene and a surfactant.
2. Background
In 2009, the Renewable Fuels Standard (RES) called for blending 11.1 billion gallons of ethanol and other biofuels into the U.S. motor fuels market to satisfied future demands. This will result in an increase in the level of corn needed by the industry and require plant capacity to be increased as well. In just the past year, the USA's annual operating capacity increased by 2.7 billion gallons, a 34% increase over 2007. This growth in production capacity was enabled by the completion, start-up, and operation of new ethanol refineries.
Ethanol, a promising biofuel from renewable resources, is produced from the starch of cereal grains (corn, sorghum, wheat, triticale, rye, malted barley, rice), tuber crops (potatoes) or by direct use of the sugar in molasses, sugar cane juice or sugar beet juice. Ethanol can also be produced by fermentation of cellulose-based material (switch grass, pine trees), but this technology has not been widely commercialized.
Eighty percent of the world ethanol is produced by Brazil and the USA. Of this, 60% is produced by yeast fermentation of corn or sugar cane juice. Ethanol production through anaerobic fermentation of a carbon source by the yeast Saccharomyces cerevisiae is one of the best known biotechnological processes and accounts for a world production of more than 35 billion liters of ethanol per year (Bayrock, 2007).
The process of ethanol production from cereals begins with the hydrolysis of starch. The hydrolysis of starch results in the conversion of amylose, a mostly linear α-D-(1-4)-glucan, and branched amylopectin, a α-D-(1-4)-glucan which has α-D-(1-6) linkages at the branch point, into fermentable sugars that subsequently are converted to ethanol by yeast (Majovic, 2006), bacteria (Dien, 2003). Bacteria are used for the production of ethanol from mostly cellulose containing material, they include Zymomonas spp., engineered E. coli, Klebsiella oxytoca, Zymomonas mobilis, Acetivibrio celluloyticus within others (Dien, 2003)
In an ethanol production system, whole corn kernel is ground and mixed with water. The mixture is then steam cooked to gelatinize starch and to decrease bacterial contamination. After this liquefaction, enzymes and yeast are added to start the fermentation process to produce ethanol.
Dry milling and wet milling are the two primary processes used to make ethanol in the United States.
In the dry milling process, the entire corn kernel or other starchy material is ground into flour and mixed with water to form a slurry. Then, enzymes are added to the mixture, which is processed in a high-temperature cooker, cooled and transferred to fermenters where yeast is added and the conversion of sugar to ethanol begins. After fermentation, the resulting mixture is transferred to distillation columns where the ethanol is separated. The solids resulting after fermentation and ethanol separation are processed to produce distiller's dried grains with solubles (DDGS) which is used for animal production, e.g. poultry, swine, and cattle feed. More than 80% of today's ethanol capacity utilizes the dry mill process (RFS, 2006).
In the wet milling process, the grain is soaked or steeped in water to facilitate the separation of the grain into its basic nutritional components, such as corn germ, fiber, gluten and starch components. After steeping, the corn slurry is processed through a series of grinders and the components separated. The gluten component is filtered and dried to produce the corn gluten meal (CGM), a high-protein product used as a feed ingredient in animal operations. The starch and any remaining water from the mash are then processed in one of three ways: Fermented into ethanol, dried and sold as dried or modified corn starch, or processed into corn syrup (RFS, 2006).
Both the wet and dry mill processes utilize only the starch portion of the corn kernel for ethanol production. The remaining protein, fat, fiber and other nutritional components remain available for use as animal feed.
In the conventional fermentation process, yeast culture is added to the starch kernel portion of the corn and incubated 72 hours to allow sufficient time for the yeast population to increase to the necessary concentration (Maye, 2006). It takes from 45 to 60 minutes for the yeast population to double. It takes many hours of such propagation to produce the quantity of yeast necessary to ferment such a large quantity of sugar solution (Maye, 2006).
A process called raw starch hydrolysis converts starch to sugar which is then fermented to ethanol, bypassing conventional starch gelatinization conditions. The enzymes used in the saccharification/fermentation are fungal alpha amylase and glucoamylase (amyloglucosidase) (Thomas, 2001). This simultaneous saccharification and fermentation allows for higher concentrations of starch to be fermented and results in higher levels of ethanol. If the sugar source is from crops such as sugar cane, sugar beets, fruit or molasses, saccharification is not necessary and fermentation can begin with the addition of yeast and water (Maye, 2006).
One of the important concerns with batch or continuous fermentation systems is the difficulty of maintaining it free from bacterial contamination. Unfortunately, the optimum atmosphere for fermentation is also optimum for bacterial growth. Contamination generally originates from harvesting of the carbohydrate material. Washing the material may help lower the contamination level (Maye, 2006).
Despite efforts to prevent contamination with cleaning and disinfecting of the saccharification tanks and continuous yeast propagation systems, biofilms can act as reservoirs of bacteria that continuously reintroduce contaminants (Bischoff, 2009).
A variety of gram positive and gram negative bacteria have been isolated from fuel ethanol fermentation including species of Lactobacillus, Pediococcus, Staphylococcus, Enterococcus, Acetobacter, Gluconobacter and Clostridium (Bischoff, 2009). Almost two thirds of the bacteria isolated were species of lactic acid bacteria, e.g. Lactobacillus (Skinner, 2007).
In a survey conducted by Skinner and Leathers (2004), 44-60% of the contaminants in the wet mill process were identified as Lactobacilli. In the dry mill process, 37 to 87% of the contaminants were identified as Lactobacilli.
Lactobacilli contamination in the range of 106 to 107 cfu/m L corn slurry can reduce ethanol yield by 1-3%. In industry, even with an active bacterial control program to control the proliferation of Lactobacilli, carbohydrate losses to Lactobacilli can make the difference between profitability and non-profitability (Bayrock, 2007). Lactobacilli not only tolerate low pH, high acidity and relatively high concentrations of ethanol, but they also multiply under conditions of alcoholic fermentation (Thomas, 2001). Bacterial contaminants compete for growth factors needed by yeast and also produce by-products that are inhibitory to yeast, particularly lactic and acetic acids.
The contamination of carbohydrate slurry during the course of alcoholic fermentation results in a) decreased ethanol yield, b) increased channeling of carbohydrates for the production of glycerol and lactic acids, c) a rapid loss of the yeast viability after exhaustion of fermentable sugars, and d) decreased proliferation of yeast in the mash in which the contaminating Lactobacilli has already grown to a high number (Thomas, 2001).
A recent survey of bacterial contaminants of corn-based plants in the US found that bacterial loads in a wet mill facility were approximately 106 cfu/mL corn slurry while those at dry-grind facilities could reach 108 cfu/mL corn slurry (Bischoff, 2007; Chang, 1997).
The presence of Lactobacillus byproducts, i.e. acetic and lactic acids, during fermentation affects yeast growth and metabolism, and it has been suggested as one of the causes of stuck, or sluggish fermentation (Thomas, 2001). If the lactic acid content of the mash approaches 0.8% and/or acetic acid concentration exceeds 0.05%, the ethanol producing yeast are stressed (Bayrock, 2007). Lactobacilli may stress yeast cells, which release nutrients, particularly amino acids and peptides that can stimulate bacterial growth (Oliva-Neto, 2004). A lactic acid concentration of 8 g/L in a beet molasses batch fermentation reduced yeast viability by 95% and alcohol production rate by 80% (Bayrock, 2001).
The presence of Lactobacillus in the ethanol fermentation can decrease ethanol yield by 44% after 4 days of pH1 controlled operation. This coincides with an increase in L. paracasei to >1010 cfu/mL and a fourfold increase in lactic acid concentration to 20 g/L. An 80% reduction in yeast density was seen with concentrations of ethanol, lactic acid and acetic acid of 70, 38 and 7.5 g/L respectively (Bayrock, 2001).
De Oliva-Neto and Yokoya (1994) evaluated the effect of bacterial contamination on a batch-fed alcoholic fermentation process. They showed that L. fermentum will strongly inhibit commercial baker's yeast in a batch-fed process. When the total acid (lactic and acetic) exceeded 4.8 g/L it interfered with yeast bud formation and viability with 6 g/L decrease in alcoholic efficiency.
Others have shown that: a) a 106 Lactobacilli/mL mash results in approx 1% v/v reduction in the final ethanol produced by yeast (Narendranath, 2004), b) challenging the fermentation system with 108 cfu/mL L. fermentum decreased ethanol yield by 27% and increased residual glucose from 6.2 to 45.5 g/L (Bischoff, 2009), c) the use of 103 cfu Lactobacilli/mL produced an 8% reduction in ethanol yield and a 3.2 fold increase in residual glucose (Bischoff 2009).
Methods to control bacteria include the addition of more yeast culture, stringent cleaning and sanitation, acid washing of yeast destined for reuse, and the use of antibiotics during fermentation (Hynes, 1997). An increased yeast inoculation rate of 3×10 7 cfu/mL. mash resulted in greater than 80% decrease in lactic acid production by L. plantarum and greater than 55% decrease in lactic acid production by L. paracasei, when mash was infected with 1×108 Lactobacilli/mL (Narendranath, 2004; Bischoff, 2009).
Various agents have been tested for control of bacterial contaminants in laboratory conditions including antiseptics such as hydrogen peroxide, potassium metabisulfite, and 3,4,4′-trichlorocarbanilide and antibiotics such as penicillin, tetracycline, monensin and virginiamycin. Penicillin and virginiamycin are commercially sold today to treat bacterial infections of fuel ethanol fermentation and some facilities use these antibiotics prophylactically (Skinner, 2004).
If no antibiotics are used, a 1 to 5% loss in ethanol yield is common. A fifty million-gallon fuel ethanol plant operating with a lactic acid level of 0.3% w/w in its distiller's beer is losing approximately 570,000 gallons of ethanol every year due to bacterial contamination (Maye, 2006). In the absence of an antibiotic, bacterial numbers increased from 1×106 cfu/mL to 6×106 cfu/m L during a 48 hour fermentation period and 5.8 mg lactic acid was produced (Hynes, 1.997).
One very effective bacterial control program involves the use of virginiamycin. Some characteristics of virginiamycin are a) at low concentrations, e.g., 0.3 to 5 ppm it is effective against a number of microorganisms including Lactobacilli, b) the microorganisms do not tend to develop resistance, c) it does not significantly inhibit the yeast, d) it is not affected by the pH or alcohol concentration, and e) it is inactivated during ethanol distillation, therefore no residue remains in the alcohol or distilled grains (Bayrock, 2007; Narendranath 2000; Hynes, 1997).
Currently, virginiamycin is the only antibiotic known to be used at the dry-grind plant (Bischoff, 2007). The recommended dose of virginiamycin in fuel ethanol fermentations is generally 0.25 to 2.0 ppm (Bischoff, 2009) but the Minimum Inhibitory Concentration (MIC) varies from 0.5 to greater than 64 ppm (Hynes, 1997).
L. fermentum could be selectively controlled by hydrogen peroxide at concentrations of 1 to 10 mM in an ethanol fermentation process (Narendranath, 2000). Lactobacillus does not have the enzyme catalase, so it cannot decompose hydrogen peroxide and therefore is unable to eliminate its toxic effect (Narendranath, 2000).
Urea hydrogen peroxide (UHP) has been used as an antiseptic for topical applications on wounds and against gingivitis and dental plaque (Narendranath, 2000) and also serves as an antibacterial during fermentation UHP not only exhibits excellent bactericidal activity against Lactobacillus but also has an important advantage of providing usable nitrogen in the form of urea for stimulating yeast growth and fermentation rates (Narendranath, 2000).
Other methods of controlling bacterial contamination include the use of sulfites. Sulfites demonstrate bactericidal activity only in the presence of oxygen and were more effective in killing facultative L. casei which possess high levels of hydrogen peroxide related enzymes, including peroxidase (Chang, 1997) Bacterial load was also decreased when the concentration of sulfite ranged from 100 to 400 mg/L but only in the presence of oxygen. This concentration did not affect yeast populations (Chang, 1997).
An agent present in the supernatant of yeast cultures reduces the growth of Lactobacilli. This compound has not yet been characterized, though it is known to be resistant to freezing, unstable at high temperatures and destroyed when held at 90° C. for 20 minutes (Oliva-Neto 2004).
Succinic acid by itself at levels of 600 mg/L reduces Lactobacillus concentrations by 78%, in the presence of ethanol that reduction is up to 96% (Oliva-Neto 2004).
A microbial adherence inhibitor in the form of fowl egg antibodies and specific to lactic acid-producing microorganisms has been developed for use in fermenters (Nash 2009).
Only laboratory studies have shown that antibodies, sulfite and peroxide products can be beneficial in controlling lactobacilli, a problem with these products is the decrease in concentration due to oxidation and decomposition of the chemicals which will require constant monitoring of the whole process of fermentation in order to maintain an effective concentration. Decreased susceptibility to virginiamycin has been observed in Lactobacilli isolated from dry-grind ethanol plants that use virginiamycin and the emergence of isolates with multi-drug resistance to both penicillin and virginiamycin has also been reported (Bischoff 2009). So alternatives to prevent decreased ethanol yield from carbohydrate fermentation are needed.